S-Nitrosogluthathione (GSNO) is a nitrosated derivateive of gluthathione that has been suggested to act as a regulator of enzymatic activity. It has been proposed that GSNO regulates enzyme activity by transferring the nitrosyl functional group to a protein cysteinyl residue via transnitrosation enzymes that contain an essential cysteine are inhibited by this process in a reversible manner. However, thiols and S-nitrosothiols react by two different and independent mechanisms. For example, GSNO and cysteine undergo transnitrosation by a reversible reaction [k(r)=80 M(-1)s(-1), k(r)= 160 M(-1)s(-1), K=0.5]. In addition GSNO and cysteine react by an irreversible reaction to form the mixed disulfide (G-SS-Cys) and reduced compounds of nitrogen (nitrous oxide, hydroxylamine ammonia) through the intermediate formation of nitroxyl (NO). This latter reaction has a rate constant of 2x10(-2) M(-1)s(-1), much slower than transnitrosation. We have shown that for creatine kinase (CK ), which contains an essential cysteinyl residue close to the active site, S-nitroso-N-acetyl pennicillamine (SNAP) predominately acts via transnitrosation to give a protein S-nitrosothiol. However, with GSNO, the predominant reaction is thiolation to give CK-SS-G and little protein S-nitrosothiol is generated. This indicates that 1) All S-Nitrosothiols do not act via the same mechanism, 2) Inhibition of protein activity by S-nitrosothiols cannot be assumed to be due to transnitrosation. 3) Cellular targets for GSNO may overlap substantially with GSSG sensitive targets and such targets may represent a common link between the cellular response to oxidative stress and to nitrosative stress.